Bioorg. Med. Chem. Lett. 37 (2021) 127840 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl Bioconjugated technetium carbonyls by transmetalation reaction with zinc derivatives Jordi Borràs a, Joan Lecina a, Julie Foster b, Roxana Kashani b, Laura Melendez-Alafort c, Jane Sosabowski b, Joan Suades a, * a Departament de Química, Edifici C, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain b Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London, United Kingdom c Istituto Oncologico Veneto IRCCS, Via Gattamelata 64, 35128 Padova, Italy A R T I C L E I N F O A B S T R A C T Keywords: The transmetalation reaction between zinc dithiocarbamates functionalized with organic groups and the cation Technetium fac-[99mTc(H2O)3(CO)3]+ has been studied as a new strategy to bind biomolecules to this radionuclide for pre­ Carbonyl paring radiopharmaceuticals with high molar activity. All complexes were obtained in high yields by heating at Radiopharmaceutical moderate temperatures and without subsequent purification. The chemical identity was ascertained by HPLC Transmetallation comparison with the homologous rhenium complexes. Stability studies in cysteine solution and serum have Rhenium shown a good stability of the coordination set fac-[99mTc(CO)3(SS)(P)]. Preliminary biological studies of the radiocomplex functionalized with D-(+)-glucosamine with carcinoma cells have been performed. Molecular imaging methods are important in medicine because they radiopharmaceuticals.7–11 The water ligands are labile and they can be provide information about the biological processes in vivo. Molecular replaced by other ligands, leading to new rhenium and technetium ra­ imaging can be applied for noninvasive monitoring of spatial-temporal diopharmaceuticals with carbonyl ligands, which exhibit high thermo­ distribution of molecular or cellular processes, for early disease detec­ dynamic stability, kinetic inertia and high in vivo stability. The above- tion and real-time monitoring of therapeutic responses.1,2 Amongst the mentioned properties make these cations ideal organometallic pre­ molecular imaging techniques, radionuclide imaging is one of the mo­ cursors for the preparation of bioconjugated compounds by the labeling dalities able to provide functional information about the disease and of biologically active molecules. track biochemical processes in vivo. Radionuclide imaging can maps the One point that should be noted about labeling reactions with Tc- biodistribution of radiopharmaceuticals using two imaging methods, 99m, is that due to the fact that the concentration of the radioactive according to the emission properties of the radionuclide used: Positron metal in the labeling reactions is very low (10-6 – 10-8 M),4–6 it is Emission Tomography (PET) or Single Photon Emission Computed To­ common to find that the concentration of the derivatized biomolecule mography (SPECT).2,3. A radiopharmaceutical is a radioactive com­ used in the reaction is several orders of magnitude higher than the pound that can be applied as imaging or therapeutic agent in nuclear concentration of the radioactive metal.12 Therefore, to avoid saturation medicine.2,3 of the receptor site, the concentration of the unlabeled derivatized Tc-99m radionuclide is the most widely used for SPECT imaging biomolecule should be as low as possible (Fig. 1). Labeled biomolecules because it is easily accessible from the 99Mo/99mTc generators, and it must have a very high molar activity (defined as the ratio between the exhibits very suitable emission properties, such as detectable gamma radioactivity of a labeled biomolecule and the total amount of the rays with a photon energy of 140 keV and an appropriate half-life (≈ 6 biomolecule in moles13,14). h).3–6 The number of publications with organometallic radiopharma­ We proposed an approach based on a transmetalation reaction as an ceuticals has increased significantly during the last 20 years due to the alternative labeling procedure that could help to achieve a high labeling development of simple and convenient preparations of the aqua ions fac- yield with an extremely low concentration of unlabeled derivatized [99mTc(H2O)3(CO)3]+ and fac-[188Re(H2O)3(CO)3]+. These cations are biomolecule in the medium.15 suitable organometallic synthons for the preparation of new Transmetalation is an essential reaction in organometallic chemistry, * Corresponding author. E-mail address: joan.suades@uab.es (J. Suades). https://doi.org/10.1016/j.bmcl.2021.127840 Received 5 November 2020; Received in revised form 20 January 2021; Accepted 26 January 2021 Available online 5 February 2021 0960-894X/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). J. Borràs et al. Bioorganic & Medicinal Chemistry Letters 37 (2021) 127840 Bio mo Biomolecule lec ule Biomol ecule Biomolecule Protein Receptor Biomolecule ec ule Biomol (CO) 3Tc [Tc(H2O)3(CO)3]+ Scheme 3. [2 + 1] Transmetalation reaction with rhenium. cule ole om Bi Biomolecule Fig. 1. Schematic representation of the competition between labeled and un­ labeled molecules towards the receptor sites. Scheme 4. Synthesis of 4(Re) complex. state + 1 (d6 configuration).15 A recent publication has applied this idea to the preparation of diaminedithiol radiopharmaceuticals.17 Reactions with Tc-99m for radiopharmaceutical uses should be performed in aqueous media, because this radiometal is prepared in saline solution and Zn-dithiocarbamates are a useful family of com­ pounds for our purposes. As mentioned above, they exhibit low solu­ bilities in water and give rise to transmetalation reactions with transition metal atoms.18 In the previous paper,15 we reported that the succinimidyl ester derivative of a zinc(II) dithiocarbamate of iso­ Scheme 1. Representation of the strategy for obtaining of potential radio­ pharmaceuticals based on transmetallation reaction. nipecotic acid (top, Scheme 2) is a valuable synthon for preparing bio­ conjugated Zn compounds (in fact, a posterior publication19 has shown the versatility and utility of this complex in the synthesis of bio­ conjugated transition metals). Thus, compounds 1–3 were reported (see scheme 2) and the viability of the transmetalation reaction between zinc and rhenium was demonstrated.15 However, the transmetalation reac­ tion in aqueous medium was only studied with rhenium in a concen­ tration nearly two orders of magnitude higher than the metal concentration commonly used in the preparation of Tc-99m radiophar­ maceuticals. In the present work, we have undertaken the study of the feasibility of the transmetalation reaction between the previously re­ ported zinc complexes and Tc-99m in water medium but using the low Tc-99m concentration obtained from 99Mo/99mTc generators, commonly used for the preparation of Tc-99m radiopharmaceuticals. Although the chemistry of technetium and rhenium shows many similar aspects, there are also notable differences as the stability of the oxida­ tion states and the kinetics of the substitution reactions,20,21 which force to design different approaches to obtain the homologous technetium complexes. Furthermore, preliminary biological studies with one of the new reported Tc-99m complexes have been performed in order to study the viability of the hypothesis that this approach could lead to radiophar­ maceuticals with an improved molar activity. Scheme 2. Preparation of bioconjugated zinc complexes 1–3. Part of this work was previously communicated22 and a recently published study23 has shown that this transmetallation reaction can be which has been used since the beginnings to prepare new compounds by applied to the preparation of metallosurfactants. the transfer of one ligand between two metals. It is still as an important In a previous publication15 we reported the synthesis of [2 + 1] fac- reaction in some fields, such as the preparation of N-heterocyclic car­ [Re(CO)3(SS)(PPh3)] complexes by means of the transmetalation reac­ benes (NHCs).16 Our proposal is to use a transmetalation reaction be­ tion using a bidentate ligand (SS = dithiocarbamate) and a monodentate tween zinc complexes of the derivatized biomolecules, which, in ligand (PPh3) (Scheme 3). general, show very low solubilities in water, with the radioactive metal In the present work, we want to increase the versatility of our to obtain high labeling yield in a medium with a very low concentration approach using other useful monodentate ligands from a radiopharma­ of the derivatized biomolecule. This transmetalation reaction is ther­ ceutical point of view, such as sodium triphenylphosphine trisulfonate modynamically favorable owing to the higher stability of bonds with the (TPPTS). Consequently, the following model rhenium complex with this {M(CO)3} fragment (Scheme 1), where M is Tc or Re in the oxidation monodentate ligand was synthetized and characterized (Scheme 4). The use of glucosamine as delivery agent of Tc-99m or other metals 2 J. Borràs et al. Bioorganic & Medicinal Chemistry Letters 37 (2021) 127840 Fig. 2. Chromatograms of each Tc-99m complex and their corresponding ho­ mologous rhenium complex. Table 1 Scheme 5. Synthesis of Tc-99 m complexes (complexes 5(Re) and 6(Re) were Results of the solubility of each zinc complex obtained by previously reported15). ICP-OES analysis. Data are expressed in ppm and molarities are shown in parenthesis. has been widely studied since 18F-FDG was discovered to be a very Zn complex Solubility efficient radiopharmaceutical for myocardial perfusion or tumor 1 11 (1.3⋅10-5M) detection.24–29 For the design of the rhenium homologue of the potential 2 3.1 (5.0⋅10-6M)15 Tc-99m radiopharmaceutical, TPPTS was chosen as a monodentate 3 0.66 (1.0⋅10-6M)15 ligand due to its water solubility and because it increases the hydro­ philicity of the complex, favoring its excretion by the renal-urinary Thus, the Tc-99m complexes are characterized by the usual methods system.30–32 The product was prepared by heating to reflux a meth­ based on comparing the HPLC retention times of the technetium com­ anol solution of [NEt4]2[ReBr3(CO)3] and complex 1, following by the plex with its homologue of rhenium that has been previously fully addition of TPPTS to obtain the expected rhenium complex. The final characterized.4–6,33 Fig. 2 shows chromatograms for each Tc-99m radi­ product was obtained with a moderate yield (56%). Two signals at 18.8 ocomplex and their rhenium homologue. As can be seen, the similar and 18.5 (minor) ppm in 31P NMR spectra certainly confirmed that the retention times between rhenium and technetium complexes confirm [2 + 1] reaction took place. This phenomenon was previously observed that the radiosynthesis took place successfully. The small differences in similar compounds with PPh3 as a monodentate ligand.15 The fact of between the UV-trace (rhenium complexes) and the gamma-trace (Tc- observing two peaks was explained by the presence of two isomers in 99m complexes) in Fig. 2 are a consequence of the different times that solution of the piperazine ring (axial, equatorial) due to the non-free the sample needs to pass through the two detectors and also to the rotation of the C–N bond of the dithiocarbamate group.15 Spectro­ different concentration of rhenium and technetium complexes in solu­ scopic (IR, 1H and 13C NMR) and spectrometric (HRMS) data, as well as tion. A relevant point is the yields obtained for all the radiosynthesis elemental analysis, were consistent with the proposed structure. The RP- performed by transmetalation with zinc complexes studied in this work, HPLC analysis showed a peak at the retention time of 11.7 min (using which are values that have been obtained without any kind of purifi­ method 3, see Supplementary Data), a piece of data that will be useful to cation. Values of 95–98% were achieved for complexes 4(Tc-99m)(98%), characterize the homologous Tc-99m complex. 5(Tc-99m)(95%), and 6(Tc-99m)(95%). These data are consistent with the All reactions with Tc-99m (Scheme 5) were performed under potential application of the transmetalation reaction in the preparation experimental conditions similar to those commonly used in the prepa­ of Tc-99m radiopharmaceuticals. ration of Tc-99m radiopharmaceuticals. Thus, in all cases, reactions are As has been pointed out in the introduction, the objective of our conducted by adding a suspension of an small amount of the appropriate approach is to achieve high chemical yields in the transmetalation re­ zinc complex (1–3, ~1mg)15 and monodentate ligand to an aqueous action using zinc complexes with a solubility as low as possible, in order solution of [99mTc(H2O)3(CO)3]+ and subsequent heating at moderate to improve the molar activity of the radiopharmaceutical. temperatures (45–65 ◦ C) for a short time (~25 min). After cooling down, Table 1 shows the water solubilities of the zinc complexes that have the suspension is centrifuged and the supernatant is ready to be analyzed been measured from aqueous saturated solution of the zinc compounds in biological studies as a potential radiopharmaceutical. It should be by inductively coupled plasma optical emission spectrometry (ICP-OES). highlighted that reaction temperatures and the short reaction times These data confirm our hypothesis that the transmetalation reaction can make this procedure compatible with the usual preparation methods of lead to the preparation of the Tc-99m complexes in high yields, in a Tc-99m radiopharmaceuticals. In addition, the fact that the synthesis is reaction medium with a very low concentration of the zinc complex. carried out under moderate temperatures makes possible to apply this Consequently, if in the previous work15 it was only possible to verify that method to a wide range of biomolecules that are denatured at higher the transmetalltion reaction was possible with rhenium, the present temperatures. study has confirmed that this approach is potentially useful for the The Tc-99m concentration in these preparations is so low that is not preparation of radiopharmaceuticals with high molar activity. possible to use standard methods to characterize the radiocomplexes. 3 J. Borràs et al. Bioorganic & Medicinal Chemistry Letters 37 (2021) 127840 18 Fig. 5. Results of binding and blocking assay for 4(Tc-99m) and F-FDG (mean ± std. dev.; n = 3). biological studies to demonstrate that this approach could be used to produce radiopharmaceuticals with an improved molar activity. The radiocompound 4(Tc-99m) was used for in vitro studies because it showed Fig. 3. Stability study of complexes 4(Tc-99m), 5(Tc-99m) and 6(Tc-99m) after in­ good stability in cysteine and serum, in addition it could be transported cubation with cysteine amino acid (RCP = Radiochemical Purity; mean ± std. into the cell via the GLUT transporters due to its glucose similar struc­ dev.; n = 3). ture. Since the GLUT-1 transporters are overexpressed in cancer, we choose to evaluate the radiolabeled compound in the human epidermoid carcinoma cell line, A431.37–38 To evaluate the transport of the potential radiopharmaceutical by GLUT-1, cell uptake experiments were carried out using the 4(Tc-99m) radiocompound, both with and without 3 mM glucose (competitor biomolecule) in parallel. This provides a measure of the efficiency and specificity of the transport, as the free glucose should saturate the uptake mechanism since the concentration is about 4 orders of magnitude higher than that of the 4(Tc-99m) complex. Thus, if uptake is occurring via GLUT-1, a significant drop in the radioactivity uptake should occur on addition of glucose. The results are shown in Fig. 5, and are compared with results obtained using 18F-FDG (the most commonly- used radiopharmaceutical for targeting the GLUT-1 transporter) as the radiotracer.39–40 It is clear that the absolute value of the uptake is much higher for 18F- FDG than 4(Tc-99m), which is not an unexpected result since 18F-FDG is a Fig. 4. Stability study of complex 4(Tc-99m) in mice serum. (RCP = Radio­ small molecule that shows very efficient transport via GLUT-1.41 chemical Purity; mean ± std. dev.; n = 3). Nevertheless, similar to 18F-FDG, 4(Tc-99m) showed a statistically sig­ nificant drop in uptake in the presence of non-radioactive glucose, A first approach to evaluate the in vivo stability of the potential ra­ suggesting that our complex could undergoes moderate uptake into diopharmaceuticals was performed by incubation of these compounds in cancer cells via the GLUT-1 transporter. However, further in vitro studies presence of cysteine, since it is one of the amino acids present in the need to be done to clearly demonstrate that 4(Tc-99m) uptake is due to a blood with high coordination ability. Therefore, radiocompounds that specific mechanism regulated by the GLUT-1 transporter. remain unaltered after some hours of incubation in solution with an It should be highlighted that our results are in accordance with excess of cysteine, demonstrated a good stability against to the ligand previously reported data in the literature, because all 99mTc- glucos­ substitution reactions. amine bioconjugates reported have unfortunately shown low uptake The radiocomplexes 4(Tc-99m), 5(Tc-99m) and 6(Tc-99m) were added to a values in tumor cells24–29,42–43. In this case the 4(Tc-99m) low uptake solution of cysteine (10-3M; molar ratio cysteine/radiocomplex was could be due to the fact that the bioactive moiety is too close to the metal 103–105) in phosphate buffered saline (PBS) for 4 h at 37 ◦ C. Aliquots center, causing steric hindrance. Thus, probably the linker should be were withdrawn at 1, 2, 3 and 4 h, and analyzed with RP-HPLC in order larger and less rigid in order to promote efficient transport, decreasing to know the radiochemical purity (RCP).34 Results are shown in Fig. 3, steric hindrance. This slight modification would be compatible with our evidencing that the coordinative set [99mTc(CO)3(SS)(P)] shows a method, and relatively easy to implement. Moreover, taking into ac­ remarkable stability in cysteine medium in the first three hours. It count that our approach does not require any purification steps, it is should be highlighted that 4(Tc-99m), which is one of the most interesting worth mentioning that, as far as we know, using this approach we have radiocomplex from a radiopharmaceutical perspective, is the most sta­ ble compound after four hours. Serum stability studies can provide additional information about Table 2 whether the studied radiocomplexes would remain unaltered in physi­ Biodistribution results of complex 4(Tc-99m) in mice. Data are presented as a ological conditions.35,36 This experiment was only performed for com­ mean from 3 animals ± SEM. plex 4(Tc-99m) because it is the most attractive radiocompound from the Organs and tissues Amount of tracer uptake (%ID/g) radiopharmaceutical point of view, since it is bioconjugated with a Tumor 0.07 ± 0.02 biologically active molecule and it is reasonably stable in cysteine me­ Intestine 20.8 ± 2.9 dium. Thus, for this experiment, compound 4(Tc-99m) was added to fresh Pancreas 0.024 ± 0.005 mouse serum and incubated at 37 ◦ C for 6 h. At time points 1, 2, 4 and 6 Spleen 0.036 ± 0.006 h, aliquots were removed and the proteins were precipitated with Kidney 0.8 ± 0.1 Liver 0.6 ± 0.3 acetonitrile. The RCP of each aliquot was measured by RP-HPLC. The Heart 0.039 ± 0.003 results showed that the radiocomplex remains almost unaltered for 4 h Lung 0.09 ± 0.02 in serum (Fig. 4). Hence, we can conclude that the radiocomplex studied Blood 0.097 ± 0.012 may be used for further in vitro and in vivo experiments. Muscle 0.019 ± 0.006 Tail 0.15 ± 0.01 As mentioned above, one of our aims was to perform preliminary 4 J. Borràs et al. Bioorganic & Medicinal Chemistry Letters 37 (2021) 127840 out a transmetalation reaction that allows the easy and straightforward bioconjugation of tricarbonyl technetium complexes with potentially high molar activity without any purification step. To the best of our knowledge, the reported value of 7.1 × 102 MBq/nmol is significantly higher than previously reported data for technetium carbonyl complexes linked to biomolecules. Despite the low water solubility of the zinc precursors (1–10 ppm), the reaction is so thermodynamically favored that it takes place with a very high yield in the presence of a phosphine ligand, which is important in order to tune the physico-chemical prop­ erties of the potential radiopharmaceutical. In addition, the reaction conditions of the transmetalation reaction are mild, which makes it possible to apply this approach to other biomolecules that cannot be stable in harsher reaction conditions. Stability studies in cysteine and serum media have provided evi­ dence of the stability of the [99mTc(CO)3(SS)(P)] coordination set in biological medium, which makes its application viable for radiophar­ maceutical purposes. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper Fig. 6. SPECT/CT image of a mouse that has been previously injected complex 4(Tc-99m). One hour post-injection (A) and four hours post-injection (B). Acknowledgments been able to reach better uptake values compared with other previously This work was supported by Spanish MINECO-FEDER grants reported Tc-99m tricarbonyl compounds bioconjugated with (BIO2015-67358-C2-2-P and CTQ2015-70371-REDT) and by General­ glucosamine.24–29,42–43 itat de Catalunya (2017SGR-795). Three female athymic mice (6 weeks old) bearing a subcutaneous We thank to Dr. Ramon Barnadas (Departament de Bioquímica, UAB) tumor (A431 cells) on the right flank were used for biodistribution for valuable helpful in the revision of the manuscript and to Dr. Imma studies. The animals were euthanized 4 h after injection of the 4(Tc-99m) Ponte (Departament de Bioquímica, UAB) for allowing us to use the complex and the main organs were harvested and weighed. The activity laboratory for handling radioactive substances. of each organ was measured using a gamma counter. Given the activity injected and the concentration of 1, we have calculated that the molar Appendix A. Supplementary material activity in this experiment was 7.1 × 102 MBq/nmol. This value is one of the highest value of molar activity reported so far for a Tc-99m com­ Supplementary data to this article can be found online at https://doi. plexes linked to biomolecules.44 The radioactivities measured in the org/10.1016/j.bmcl.2021.127840. individual tissues were expressed as the percentage of the total injected radioactivity, per gram of tissue (Table 2). It should be highlighted that the radiocomplex was highly accumulated in the intestines, liver and References kidneys. In general terms, these results are in agreement with previously 1 Melendez-Alafort L, Muzzio PC, Rosato A. 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